Ventilator-associated pneumonia (VAP) is a serious complication in mechanically ventilated children, increasing risk of mortality and morbidities including prolonged intubation and intensive care unit stays. Limited understanding of the microbial and host factors associated with VAP has precluded development of truly effective prevention and treatment strategies. Recent evidence has revealed that the lungs, previously considered sterile, contain microbial populations. These endogenous bacteria are likely critical regulators of both pathogen behavior and host responses in the airways, modulating the virulence of potential pathogens. In addition, respiratory failure secondary to viral lower respiratory tract infections is common among children and is often complicated by bacterial co-infections, including VAP. Yet, standard bacterial and viral culture methodologies are inefficient and insensitive for identifying the full spectrum of airway microorganisms associated with VAP. New methodologies are needed to better understand the pathogenesis of VAP. Study of the microbiome, the genomic content of the microbiota (the organisms living in an environment), is a novel approach to describe the complexities of the airway microbiota. Culture-independent molecular techniques, using nucleic acids isolated from routinely collected respiratory samples, can provide sensitive quantification of the bacterial and viral constituents of the microbiome elucidating ecological changes that may precede development of VAP. Viral infections may alter the endogenous flora and may also activate host immune suppressive responses that lead to less effective control of bacterial populations and subsequent bacterial infection. However, there has not been a systematic evaluation of virome-bacterial microbiome interactions in this population. We hypothesize that specific ecological patterns of the airway microbiome precede VAP and that the interaction of viral infection, bacterial populations, and the balance between host immune activation and immune suppression play crucial roles in determining whether a given patient develops VAP. We propose to investigate this hypothesis with a longitudinal (daily) examination of the airway microbiome in high risk mechanically ventilated children together with simultaneous proteomic assessments of the host immune response. Aim 1 of this proposal will characterize the changes in the airway microbiome of mechanically ventilated patients that precede the development of VAP. Aim 2 will determine whether respiratory viral infections interact with the airway microbiome to promote changes in the microbiota associated with VAP. Aim 3 will explore the molecular and immunologic mechanisms by which these viral-bacterial interactions promote lung infection, and will test the specific hypothesis that virally-induced hos immune suppressive pathways are preferentially up-regulated in those patients who develop VAP. This project will provide insight into the pathogenesis of VAP, uncover unique biomarkers of disease, and identify potential targets for novel prevention and treatment strategies. 1